All referenced publications and patent applications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed inventions, or that any publication specifically or implicitly referenced is prior alt.
Increasingly, greater attention is being focused on the production and use of larger and more complex protein molecules as therapeutic agents. Examples of such therapeutic proteins include antigens used in vaccinations to induce immune responses and antibodies.
Plants have great potential as hosts for the production of mammalian therapeutic proteins including multimeric proteins such as antibodies. See, for example, Hiatt, A. et al., Nature 342(6245):76-78 (1989); Hein et al., Biotechnol. Prog. 7(5):455-461 (1991); Hiatt, A., and Ma, J. K., FEBS Lett 307(1):71-5 (1992); Ma et al., Eur. J. Immunol. 24:131-138 (1994); Ma et al., TIBTECH 13:522-527 (1995); Zeitlin et al., Nature Biotechnology 16(1361-1364 (1998); Ma, H. K.-C et al. Nature Medicine 4(5):601-606 (1998); Miele, L., Trends Biotechnol. 15: 45-50 (1997); Khoudi et al., Biotechnology and Bioengineering 64(2):135-143 (1999); and, Hood, E. E. & Jilka, J. M., Curr. Opin. Biotechnol. 10: 382-386 (1999). The benefits of using plants for antibody production include large scale production, reduced costs for production, maintenance and delivery as well as eliminating the risk of the resultant product containing possibly harmful contaminants such as viruses or prions that are pathogenic to humans and other mammals. Plants, like other heterologous expression systems including mammalian cells, bacteria, yeast, and insects, exhibit differences in glycosylation. See, for example, Ma et al., Science 268:716-719 (1995); Jenkins et al., Nat. Biotechnol. 14: 975-981 (1996); and Lerouge et al., Plant Mol. Biol. 38: 31-48 (1998).
In plants, as in other eukaryotes, most of the soluble and membrane bound proteins that are synthesized on polyribosomes associated with the endoplasmic reticulum (ER) are glycoproteins, including those proteins which will later be exported to the Golgi apparatus, lysosomes, plasma membrane or extracellular matrix. The glycans attached to glycoproteins contain a variety of sugar residues linked in linear or branched structures that can assume many different conformations. These glycans can play a fundamental role in promoting correct protein folding and assembly and, as a consequence, enhance protein stability. They may also contain targeting information, or may be directly involved in protein recognition (Maia et al., Genetics and Molecular Biology 24: 231-234 (2001)). The three main posttranslational modifications of proteins that involve carbohydrates are N- and O-linked glycosylation and the insertion of glycosyl phosphatidyl inositol anchors.
The N-linked glycosylation mechanisms in mammalian and plant systems have been conserved during evolution. However, differences are observed in the final steps of oligosaccharide trimming and glycan modification in the Golgi apparatus. In contrast to bacteria, having no N-linked glycans, and yeast, having polymannose glycans, plants produce glycoprotein multimers with complex N-linked glycans having a core substituted by two N-acetylglucosamine (GlcNAc) residues. These glycoprotein multimers are also observed in mammals. See, for example, Kornfeld and Kornfeld, Ann. Rev. Biochem. 54:631 (1985). Plant and animal glycopolypeptide multimers contain different terminal carbohydrates that are directly linked to the outer branches of the oligosaccharides present. Animal glycopolypeptide multimers, including mammalian glycopolypeptide multimers, have sialic acid present as a terminal carbohydrate residue, while plant glycopolypeptide multimers do not. The terminal core is substituted by β1,2-linked xylose (Xyl) and α1,3-linked core fucose (Fuc) instead of α1,6-linked core fucose as occur in mammals. Furthermore, plant glycoprotein multimers lack the characteristic galactose (Gal)- and sialic acid-containing complex N-glycans (N-acetylneuraminic-α2-6/3Galβ1-4) found in mammals. See, for example, Sturm et al., J. Biol. Chem: 262:13392 (1987). A murine monoclonal antibody produced in transgenic plants with plant-specific glycans was found not to be immunogenic in mice (Chargelegue et al., Transgenic Research 9:187-194 (2000)).
Antibodies have conserved N-linked glycosylation of the Fc region of each of the two heavy chains. Human IgA antibodies have O-linked oligosaccharides in their hinge portion and two N-linked carbohydrate chains; one occurring on an asparagine (Asn) residue in the CH2 region of the heavy chain and the other on an Asn residue in the tailpiece region. See, for example, Baenzinger, J. and Kornfield, S. J., Biol. Chem. 249:7260-7269 (1974); and Torano et al., PNAS 74:2301-2305 (1997). Fucosylation of the IgA isolated from human serum occurs only on the Asn in the tailpiece region (Tanaka et al., Glycoconj. J. 10: 995-1000 (1998)).
Hiatt et al. have produced transgenic plants expressing nucleotide sequences encoding individual or assembled immunoglobulin heavy- and light-chain immunoglobulin polypeptides. Each immunoglobulin product was expressed as a proprotein containing a leader sequence forming a sequence which directs the protein into the endosecretory pathway allowing correct assembly and glycosylation of the antibody molecule. The leader sequence is cleaved from the mature protein. See, for example, U.S. Pat. Nos. 5,202,422; 5,639,947 and 6,417,429. Methods for the coordinated expression and production of secretory immunoglobulins containing heavy chain, light chain, J chain and secretory component polypeptides which are assembled into functional antibodies have been disclosed. See, for example, U.S. Pat. Nos. 5,959,177; 6,046,037 and 6,303,341. Each of the U.S. patents cited herein is incorporated by reference in its entirety. A murine immunoglobulin transmembrane sequence was used for plasma membrane targeting of recombinant immunoglobulin chains in plants (Vine et al., Plant Molecular Biology 45:159-167 (2001)).